The invention discloses a detection kit and a detection method of circulating tumor cells. The kit comprises a PBS buffer solution, a density gradient separation medium, a red blood cell lysate, a CD45 immunomagnetic bead, an incubation solution, a fixing solution, a permeabilizing solution, a confining liquid, an antifade mounting medium, an antibody dilution solution and a fluorescent antibody concentrate, and the fluorescent antibody concentrate contains a fluorescent dye pan-CK-AF488, a fluorescent dye CD45-PE and a cell nucleus dye Hoechst33342. The detection method comprises the following steps: S10, centrifuging a blood sample, and separating and enriching the CTCs; and S20, carrying out dyeing identification on the enriched CTCs through the cell nucleus dye pan-CK-AF488, the fluorescent dye CD45-PE and Hoechst33342 fluorescence antibodies, wherein the detection is carried out for the purpose of non-treatment. When the detection kit and the detection method are adopted to perform detection, the detection result is accurate and reliable.